Cofactor regeneration system

ABSTRACT

The present invention relates to cofactor regeneration systems, components and uses thereof and methods for generating and regenerating cofactors. The cofactor regeneration system comprises a first electron transfer component selected from a polypeptide comprising a NADH:acceptor oxido-reductase or NADPH:acceptor oxido-reductase, a second electron transfer component selected from a hydrogenase moiety and/or non-biological nanoparticles and an electronically conducting surface. The first and second electron transfer components are immobilised on the electrically conducting surface, and the first and second electron transfer components do not occur together in nature as an enzyme complex.

This application is a Continuation of U.S. patent application Ser. No.14/349,126, filed 2 Apr. 2014, which is a National Stage Application ofPCT/GB2012/052451, filed 3 Oct. 2012, which claims benefit of Serial No.1116971.1, filed 3 Oct. 2011 in Great Britain and which applications areincorporated herein by reference. To the extent appropriate, a claim ofpriority is made to each of the above disclosed applications.

The work leading to this invention has received funding from theEuropean Research Council under the European Union's Seventh FrameworkProgramme (FP7/2007-2013)/ERC grant agreement No. 258600.10.

The present invention relates to a cofactor regeneration system,components and uses thereof, products comprising said cofactorregeneration system and components thereof, as well as methods forcofactor generation and regeneration.

Cofactors are non-protein chemical compounds that play an essential rolein many enzyme catalysed biochemical reactions. Cofactors act totransfer chemical groups between enzymes. Nicotinamide adeninedinucleotide (NAD⁺), and nicotinamide adenine dinucleotide phosphate(NADP⁺) and the reduced forms of said molecules (NADH and NADPH,respectively) are biological cofactors which play a central role in themetabolism of cells acting as electron transfer agents. The oxidizedforms NAD⁺ and NADP⁺ act as electron acceptors, becoming reduced in theprocess. NADH and NADPH, in turn, can act as reducing agents, becomingoxidized in the process.

Enzymes are commonly used as biocatalysts in the chemical andpharmaceutical industries. Redox enzymes—those that mediate oxidation orreduction reactions—form a significant subset of enzymes that are usefulin industrial applications. However, most redox enzymes are dependent onexpensive cofactors such as NADPH (approx. $500 for 0.75 g). To date,the use of NAD(P)H-dependent catalysts has been severely limited by theabsence of industrially useful methods for recycling NAD(P)H. Currently,NADH is often regenerated using a formate dehydrogenase system. Thisprocess produces CO₂ which strongly affects the pH of the reactionsolution. The formate dehydrogenase system is characterised by a lowturnover frequency and a limited half-life. Also, formate (the substratefor cofactor regeneration) contaminates the product of the coupledenzyme reaction. NADPH is currently often regenerated using a glucosedehydrogenase system in which glucose and its oxidized form contaminatethe product of the coupled enzyme system.

Electrochemical regeneration of cofactors at conventional electrodesrequires a large overpotential (meaning loss of energy). Some modifiedelectrodes have been reported, e.g. with poly(Neutral Red), butmodifiers may be toxic or damaging to enzymes. Another majordisadvantage is that bio-inactive forms (e.g. dimers) of NAD⁺/NADH maybe generated in the electrode reaction.

There is, therefore, a need to provide an alternative and/or improvedcofactor regeneration system.

The present invention solves one or more of the above mentionedproblems.

In one aspect, the invention provides a cofactor regeneration systemcomprising or consisting of:

-   -   i) a first electron transfer component selected from one or more        polypeptides comprising a NADH:acceptor oxido-reductase or a        NADPH:acceptor oxido-reductase,    -   ii) a second electron transfer component selected from a        hydrogenase moiety and/or non-biological nanoparticles; and,    -   iii) an electronically conducting surface;        wherein the first and second electron transfer components are        immobilised on the electrically conducting surface, and wherein        the first and second electron transfer components do not occur        together in nature as an enzyme complex.

The invention advantageously provides a highly efficient, rapid and/orrobust cofactor regeneration system for biological cofactors. Saidsystem provides a welcome replacement for the expensive and/orinefficient systems presently being used. As discussed above, extantmethods of electrochemical cofactor regeneration require a fairly largeoverpotential (meaning loss of energy) to operate. The present inventionadvantageously utilizes catalysts that work at minimal, or undetectable,overpotential and is energetically efficient.

In addition, the invention advantageously provides a cofactorregeneration system which is modular in structure. Thus, the system canbe tuned (by choice of different components of the system) for aspecific application and/or condition(s). This provides a great deal offlexibility and allows optimization of the cofactor regeneration systemdepending on the application/conditions. For example, an oxygen(O₂)—tolerant hydrogenase may be selected as the second electrontransfer component if the cofactor regeneration system is used to supplycofactors to enzymes requiring O₂, such as cytochrome P450mono-oxygenases. In contrast, extant systems employing formatedehydrogenase (FDH) and/or glucose dehydrogenase (GDH) are limited tospecific operating condition requirements (e.g. a narrow pH range).

Another advantage of the present invention is the ability to employ H₂as the electron donor and/or H⁺ as the electron sink. The systemtherefore does not require the addition of other soluble reagents orproducts, which are hard to separate after cofactor regeneration hastaken place.

Examples of cofactors embraced by the present invention includenicotinamide adenine dinucleotide (NAD⁺) and the reduced form of NAD⁺,namely NADH, as well as nicotinamide adenine dinucleotide phosphate(NADP⁺) and the reduced form of NADP⁺, namely NADPH.

In one embodiment, said first electron transfer component comprises orconsists of a NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase. Said enzymes catalyze the oxidation of NADH and/or NADPHand/or the reduction of NAD⁺ and/or NADP⁺—the enzyme may act as anoxidizing agent or as a reducing agent, depending on the reactionconditions. By way of example, the ratio of NAD⁺ to NADH and/or theratio of NADP⁺ to NADPH may influence the direction of the reaction i.e.whether the enzyme acts as a reducing agent or oxidizing agent. In oneembodiment, the second electron transfer component comprises or consistsof a hydrogenase in which case, the H₂ concentration may also influencethe way the reaction proceeds. Suitable reaction conditions forregeneration of each of the above-mentioned cofactors are describedbelow. Thus, in one embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase may act as an oxidizing agent and/or areducing agent.

In one embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 20% (such as at least 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%,88%, 90% 92%, 94%, 96%, 98%, 99% or 100%) sequence identity to the aminoacid sequence of Ralstonia eutropha diaphorase HoxF (SEQ ID NO: 1)and/or an amino acid sequence having at least 20% (such as at least 25%,30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 88%, 90% 92%, 94%, 96%, 98%, 99%or 100%) sequence identity to the amino acid sequence of Ralstoniaeutropha diaphorase HoxU (SEQ ID NO: 2).

In one embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 20% (such as at least 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%,88%, 90% 92%, 94%, 96%, 98%, 99% or 100%) sequence identity to the aminoacid sequence of Ralstonia eutropha diaphorase HoxF (SEQ ID NO: 1)and/or an amino acid sequence having at least 20% (such as at least 25%,30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 88%, 90% 92%, 94%, 96%, 98%, 99%or 100%) sequence identity to the amino acid sequence of Ralstoniaeutropha diaphorase HoxU (SEQ ID NO: 2) and/or an amino acid sequencehaving at least 20% (such as at least 25%, 30%, 35%, 40%, 50%, 60%, 70%,80%, 85%, 88%, 90% 92%, 94%, 96%, 98%, 99% or 100%) sequence identity tothe amino acid sequence of Ralstonia eutropha diaphorase HoxI (SEQ IDNO: 3).

In one embodiment, the NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase may comprise or consist of flavoprotein (Fp) subcomplexof Complex I of Bos taurus (SEQ ID NO: 4 and/or SEQ ID NO: 5). Thus, inone embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of the 51 kDa proteinof Bos taurus Complex I (SEQ ID NO: 4) and/or an amino acid sequencehaving at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%,99% or 100%) sequence identity to the amino acid sequence of the 24 kDasubcomplex of Bos taurus Complex I (SEQ ID NO: 5).

In another embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase comprises or consists of an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofR. eutropha NAD⁺-dependent formate dehydrogenase diaphorase moiety FdsB(SEQ ID NO: 6) and/or an amino acid sequence having at least 70% (suchas at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequenceidentity to the amino acid sequence of the R. eutropha NAD⁺-dependentformate dehydrogenase diaphorase moiety FdsG (SEQ ID NO: 7).

In another embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase comprises or consists of an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofthe NADPH oxidoreductase moiety from Pyrococcus furiosus solublehydrogenase I gamma subunit (SEQ ID NO: 8) and/or an amino acid sequencehaving at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%,99% or 100%) sequence identity to the amino acid sequence of the NADPHoxidoreductase moiety from Pyrococcus furiosus soluble hydrogenase Ibeta subunit (SEQ ID NO: 9).

In another embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase comprises or consists of an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofthe NADPH oxidoreductase moiety from Pyrococcus furiosus solublehydrogenase II gamma subunit (SEQ ID NO: 10) and/or an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofthe NADPH oxidoreductase moiety from Pyrococcus furiosus solublehydrogenase II beta subunit (SEQ ID NO: 11).

In one embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of the diaphorasemoiety of Rhodococcus opacus soluble hydrogenase HoxF (SEQ ID NO: 12),and/or an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of the diaphorase moiety of Rhodococcus opacussoluble hydrogenase HoxU (SEQ ID NO: 13).

In another embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase comprises or consists of an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofthe diaphorase moiety of Allochromatium vinosum soluble hydrogenase HoxF(SEQ ID NO: 14), and/or an amino acid sequence having at least 70% (suchas at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequenceidentity to the amino acid sequence of the diaphorase moiety ofAllochromatium vinosum soluble hydrogenase HoxU (SEQ ID NO: 15).

In another embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase comprises or consists of an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofthe diaphorase moiety of Thiocapsa roseopersicina Hox1F (SEQ ID NO: 16),and/or an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of the diaphorase moiety of Thiocapsa roseopersicinasoluble hydrogenase Hox1U (SEQ ID NO: 17).

In one embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of the diaphorasemoiety of Thiocapsa roseopersicina Hox2F (SEQ ID NO: 18), and/or anamino acid sequence having at least 70% (such as at least 75%, 80%, 85%,90%, 95%, 96%, 98%, 99% or 100%) sequence identity to the amino acidsequence of the diaphorase moiety of Thiocapsa roseopersicina solublehydrogenase Hox2U (SEQ ID NO: 19).

In another embodiment, said NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase comprises or consists of an amino acidsequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%,96%, 98%, 99% or 100%) sequence identity to the amino acid sequence ofthe diaphorase moiety of Synechocystis sp. PCC 6803 HoxF (SEQ ID NO:20), and/or an amino acid sequence having at least 70% (such as at least75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of the diaphorase moiety of Synechocystis sp. PCC6803 HoxU (SEQ ID NO: 21).

In one embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of the diaphorasemoiety of Synechococcus elongates PCC 6301 HoxF (SEQ ID NO: 22), and/oran amino acid sequence having at least 70% (such as at least 75%, 80%,85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to the aminoacid sequence of the diaphorase moiety of Synechococcus elongates PCC6301 HoxU (SEQ ID NO: 23).

In one embodiment, said NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductase comprises or consists of an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of the diaphorasemoiety of Rhodobacter capsulatus SB1003 formate dehydrogenase betasubunit FdsB (SEQ ID NO: 66), and/or an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of the diaphorasemoiety of Rhodobacter capsulatus SB1003 formate dehydrogenase gammasubunit FdsG (SEQ ID NO: 67)

Conventional methods for determining amino acid sequence identity arediscussed in more detail later in the specification.

In one embodiment, the cofactor regeneration system comprises one ormore (such as two, three, four, five, six, seven, eight, nine, or ten ormore) different NADH:acceptor oxido-reductase or NADPH:acceptoroxidoreductases. All of the above mentioned NADH:acceptoroxido-reductases or NADPH:acceptor oxidoreductases are suitable in thisregard. Any combination of the above-mentioned NADH:acceptoroxido-reductases or NADPH:acceptor oxidoreductases may be employed inthe first electron transfer component of the present invention.

The first electron transfer component of the present invention maycomprise or consist of one or more (such as two, three, four, five, six,seven or more) polypeptides in addition to the NADH:acceptoroxido-reductase or NADPH:acceptor oxidoreductase. In one embodiment, thefirst electron transfer component further comprises or consists of apolypeptide having at least 70% (such as at least 75%, 80%, 85%, 90%,95%, 96%, 98%, 99% or 100%) sequence identity to the amino acid sequenceof Ralstonia eutropha soluble hydrogenase moiety HoxHY (SEQ ID NOs: 24and/or 25). In one embodiment said HoxH sequence (SEQ ID NO: 24) isselected from Ralstonia eutropha soluble hydrogenase moiety variantHoxH_I64A (SEQ ID NO: 68). Said variant contains a substitution in theactive site of the enzyme, which renders it non-functional. Withoutwishing to be bound by theory, the present inventors believe that thepresence of a HoxHY component in the first electron transfer componentincreases the stability of the NADH:acceptor oxido-reductase orNADPH:acceptor oxidoreductase leading to increased efficiency/activityof the system. Additional methods for increasing the stability ofproteins/protein complexes are known in the art, and may be routinelyemployed by a skilled person in connection with the present invention.

In one embodiment, the first electron transfer component of the presentinvention may comprise or consist of a HoxHYFU tetramer, such as theHoxHYFU tetramer of Ralstonia eutropha (i.e. SEQ ID NOs: 24, 25, 1, 2).Thus, in one embodiment, the first electron component of the presentinvention comprises or consists of an amino acid sequence having atleast 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or100%) sequence identity to the amino acid sequence of a Ralstoniaeutropha HoxHYFU tetramer (SEQ ID NOs: 24, 25, 1, 2).

The invention also provides for individual components, such as the firstand second electron transfer components, and an electronicallyconducting surface for use in the cofactor regeneration system of theinvention.

Thus, in one aspect, the invention provides a Ralstonia eutrophadiaphorase variant, wherein said diaphorase variant comprises orconsists of an amino acid sequence having at least 70% (such as at least75%, 80%, 85%, 90%, 95%, 96%, 98%) and less than 100% (such as less than99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%) sequence identity to theamino acid sequence of Ralstonia eutropha diaphorase HoxF (SEQ ID NO:1), and/or an amino acid sequence having at least 70% (such as at least75%, 80%, 85%, 90%, 95%, 96%, 98%) and less than 100% (such as less than99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%) sequence identity to theamino acid sequence of Ralstonia eutropha diaphorase HoxU (SEQ ID NO:2), and/or an amino acid sequence having at least 70% (such as at least75%, 80%, 85%, 90%, 95%, 96%, 98%) and less than 100% (such as less than99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%) sequence identity to theamino acid sequence of Ralstonia eutropha diaphorase HoxI (SEQ ID NO:3).

In one embodiment, the cofactor regeneration system of the presentinvention comprises the Ralstonia eutropha HoxF diaphorase variantsdescribed herein. In particular, the first electron transfer componentof the cofactor regeneration system described herein embraces thediaphorase variants of the present invention.

In one embodiment, the diaphorase variant has an increased catalyticactivity for NAD⁺ and/or NADP⁺ reduction and/or NADH and/or NADPHoxidation compared to Ralstonia eutropha diaphorase comprising orconsisting of SEQ ID NO:1 and/or SEQ ID NO: 2 and/or SEQ ID NO: 3. Inone embodiment, catalytic activity may embrace K_(M) and/or k_(cat). Inone embodiment, increased catalytic activity embraces increasedaffinity, and/or reduction and/or oxidation capacity for NAD⁺, NADP⁺,NADH and/or NADPH.

In one embodiment, the diaphorase variant has an increased catalyticactivity (k_(cat) and/or K_(M)) for NADP⁺ reduction and/or NADPHoxidation compared to Ralstonia eutropha diaphorase comprising orconsisting of SEQ ID NO:1 and/or SEQ ID NO: 2 and/or SEQ ID NO: 3. Inone embodiment, the catalytic activity of said diaphorase for NADP⁺and/or NADPH is increased by a factor of at least 5 (such as at least 5,10, 20, 30, 40, 50, 100, 200, 300, 400, 600, 800, 1000, 5000, 10000 or100000). In one embodiment, the catalytic activity of said diaphorasefor NADP⁺ and/or NADPH is increased by a factor of between 5-100000.

In one embodiment, the diaphorase variant has an increased catalyticactivity (k_(cat) and/or K_(M)) for NAD⁺ reduction and/or NADH oxidationcompared to Ralstonia eutropha diaphorase comprising or consisting ofSEQ ID NO:1 and/or SEQ ID NO: 2 and/or SEQ ID NO: 3. In one embodiment,the catalytic activity of said diaphorase for NAD⁺ and/or NADH isincreased by a factor of at least 5 (such as at least 5, 10, 20, 30, 40,50, 100, 200, 300, 400, 600, 800, 1000, 5000, 10000 or 100000). In oneembodiment, the catalytic efficiency of said diaphorase for NAD⁺ and/orNADH is increased by a factor of between 5-100000.

The Michaelis constant K_(M) is a standard means of characterising anenzyme's affinity for a substrate. K_(M) represents the concentration ofsubstrate at which the enzyme has half of its maximum activity. In otherwords, a low K_(M) indicates that the enzyme reaches half of its maximumactivity at low levels of substrate. It is understood by those skilledin the art, that the K_(M) value decreases as affinity increases. Thus,an increase in affinity is characterised by a decrease in the K_(M)value. In one embodiment, the K_(M) of said diaphorase variant for NADP⁺and/or NAD⁺ and/or NADPH and/or NADH is reduced compared to thewild-type Ralstonia eutropha diaphorase comprising or consisting of SEQID NO:1 and/or SEQ ID NO: 2 and/or SEQ ID NO: 3.

Methods for measuring affinity (K_(M)) are routine to those skilled inthe art. Briefly, affinity of the diaphorase for NADH and/or NADPH canbe measured under anaerobic conditions at 30° C. in 50 mM Tris-HClbuffer, pH 8.0, containing 1 mM NADH or 1 mM NADPH, 5 mM benzyl viologen(oxidized), 90 μM dithionite, and 10 to 50 μmol of enzyme. Theabsorption is monitored spectrophotometrically at 578 nm (ε=8.9 mM⁻¹cm⁻¹ for benzyl viologen). Individual data points of the activitymeasurements are used for the determination of K_(M) by non linearregression. An electrochemical method can be used to determine K_(M).Briefly, the diaphorase moiety is adsorbed onto a pyrolytic graphiteelectrode which is immersed in an electrochemical cell solutioncontaining buffered electrolyte (eg 50 mM phosphate at pH 7.0). Theelectrode is held at a constant potential of −412 mV while theconcentration of NAD⁺ or NADP⁺ is increased by injections into thesolution. Since the electrocatalytic current magnitude recorded at theelectrode is directly proportional to catalytic activity of theimmobilised enzyme film, a plot of (substrate concentration)/(currentmagnitude) vs (substrate concentration) is analogous to a Hanes or Woolfplot of (substrate concentration)/(activity) vs (substrateconcentration), and the intercept on the (substrate concentration) axisis equal to (−K_(M)). k_(cat) is the catalytic conversion of productunder optimum conditions with substrate saturated enzyme. [L.Lauterbach, Z. Idris, K. A. Vincent, O. Lenz “Catalytic properties ofthe isolated diaphorase fragment of the NAD⁺-reducing [NiFe]-hydrogenasefrom Ralstonia eutropha” PLoS ONE doi:10.1371/journal.pone.0025939.]

An improved affinity (lower K_(M)) of the diaphorase variant for NADPHand/or NADP⁺ indicates an improved catalytic activity foroxidation/reduction of NADPH/NADP⁺. Thus, in one embodiment, referenceherein to increased or improved affinity also embraces an increased orimproved oxidation/reduction activity. Thus, in one embodiment thediaphorase variant has an increased and/or improved oxidation/reductionactivity when compared to wild-type Ralstonia eutropha diaphorasecomprising or consisting of SEQ ID NO:1 and/or SEQ ID NO: 2 and/or SEQID NO:3.

The turnover number k_(cat) gives a measure of the number of substratemolecules turned over per enzyme moiety per second. An improved turnovernumber k_(cat) of the diaphorase variants for NADP⁺ and/or NAD⁺ and/orNADPH and/or NADH indicates an increased frequency for oxidation ofNADPH and/or NADH and/or reduction of NAD⁺ and/or NADP⁺. In oneembodiment, the diaphorase variant has an increased turnover number(k_(cat)) for NAD⁺, NADP⁺, NADH and/or NADPH compared to Ralstoniaeutropha diaphorase comprising or consisting of SEQ ID NO:1 and/or SEQID NO: 2 and/or SEQ ID NO:3.

An improved turnover number (k_(cat)) of the diaphorase variant forNADPH and/or NADP⁺ indicates an improved catalytic activity foroxidation/reduction of NADPH/NADP⁺. Thus, in one embodiment, referenceherein to increased turnover number k_(cat) also embraces an increasedor improved oxidation/reduction capacity. Thus, in one embodiment thediaphorase variant has an increased and/or improved oxidation/reductioncapacity when compared to wild-type Ralstonia eutropha diaphorasecomprising or consisting of SEQ ID NO:1 and/or SEQ ID NO: 2 and/or SEQID NO:3.

An improved turnover number (k_(cat)) of the diaphorase variant for NADHand/or NAD⁺ indicates an improved catalytic activity foroxidation/reduction of NADH/NAD⁺. Thus, in one embodiment, referenceherein to increased turnover number k_(cat) also embraces an increasedor improved oxidation/reduction capacity. Thus, in one embodiment thediaphorase variant has an increased and/or improved oxidation/reductioncapacity when compared to wild-type Ralstonia eutropha diaphorasecomprising or consisting of SEQ ID NO:1 and/or SEQ ID NO: 2 and/or SEQID NO:3.

In one embodiment, said diaphorase variant comprises or consists of anamino acid sequence having at least 70% (such as at least 75%, 80%, 85%,90%, 95%, 96%, 98%, 99% or 100%) sequence identity to the amino acidsequence selected from the group consisting of SEQ ID NOs: 54-65==and/or an amino acid having at least 70% (such as at least 75%, 80%,85%, 90%, 95%, 96%, 98%, 99%, 100%) sequence identity to the amino acidsequence of Ralstonia eutropha diaphorase HoxU (SEQ ID NO: 2), and/or anamino acid having at least 70% (such as at least 75%, 80%, 85%, 90%,95%, 96%, 98%, 99%, 100%) sequence identity to the amino acid sequenceof Ralstonia eutropha diaphorase HoxI (SEQ ID NO: 3).

In one embodiment, said diaphorase variant comprises or consists of anamino acid sequence which differs from the amino acid sequence of SEQ IDNO:1 at position 326 by having an amino acid selected from the groupconsisting of K (lysine), S (serine), A (alanine), N (asparagine), R(arginine) or H (histidine). In one embodiment, the amino acid is K(lysine), and this diaphorase variant corresponds to SEQ ID NO: 54.

In another embodiment, said diaphorase variant comprises or consists ofan amino acid sequence which differs from the amino acid sequence of SEQID NO:1 at position 401 by having an amino acid selected from the groupconsisting of K (lysine), S (serine), A (alanine), N (asparagine) R(arginine) or H (histidine). In one embodiment, the amino acid is K(lysine), and this diaphorase variant corresponds to SEQ ID NO: 55.

In another embodiment, said diaphorase variant comprises or consists ofan amino acid sequence which differs from the amino acid sequence of SEQID NO:1 at position 467 by having an amino acid selected from the groupconsisting of S (serine), K (lysine), A (alanine), N (asparagine), R(arginine) or H (histidine). In one embodiment, the amino acid is S(serine), and this diaphorase variant corresponds to SEQ ID NO: 56.

In another embodiment, said diaphorase variant comprises or consists ofan amino acid sequence which differs from the amino acid sequence of SEQID NO:1 at position 340 by having an amino acid selected from the groupconsisting of A (alanine), K (lysine), S (serine), N (asparagine), R(arginine) or H (histidine). In one embodiment, the amino acid is A(alanine), and this diaphorase variant corresponds to SEQ ID NO: 57.

In another embodiment, said diaphorase variant comprises or consists ofan amino acid sequence which differs from the amino acid sequence of SEQID NO:1 at position 341 by having an amino acid selected from the groupconsisting of A (alanine), K (lysine), S (serine), N (asparagine), R(arginine) or H (histidine). In one embodiment, the amino acid is A(alanine), and this diaphorase variant corresponds to SEQ ID NO: 58. Inanother embodiment, the amino acid is H (histidine), and this diaphorasevariant corresponds to SEQ ID NO: 65.

In one embodiment, the diaphorase variant of the present inventioncomprises or consists of an amino acid sequence which differs from theamino acid sequence of SEQ ID NO:1 at one or more (such as 2, 3, 4, 5,or 6) amino acid positions. In other words, said amino acid sequence mayhave mutations (e.g. substitutions and/or deletions), such as one ormore (such as 2, 3, 4, 5, or 6) of the above-mentioned amino acidmutations.

By way of example, SEQ ID NO 59 differs from the amino acid sequence ofSEQ ID NO:1 at positions 340 and 341 by having A (alanine) amino acidsubstitutions at said positions. SEQ ID NO 60 has amino acidsubstitutions at positions 340 (alanine) and 401 (lysine). SEQ ID NO 61has amino acid substitutions at positions 326 (lysine) and 401 (lysine).SEQ ID NO 62 has amino acid substitutions at positions 467 (serine) and401 (lysine). SEQ ID NO 63 has amino acid substitutions at positions 340(asparagine) and 467 (serine). SEQ ID NO 64 has amino acid substitutionsat positions 341 (alanine) and 467 (serine).

The above-mentioned variants advantageously have a greater affinityand/or turnover number for NADP⁺/NADPH and therefore have an improvedNADP⁺/NADPH catalytic activity compared to Ralstonia eutropha diaphoraseHoxF and/or HoxU and/or HoxI (SEQ ID NO: 1 and/or SEQ ID NO:2 and/or SEQID NO:3). Other diaphorase variants embraced by the present inventioninclude variants comprising amino acid substitutions and/or deletionswhich serve to decrease the negative charges surrounding the active siteand/or increase the positive charges surrounding the active site.Without wishing to be bound by theory, the Inventors believe that suchvariants advantageously are able to accommodate the additionalnegatively charged phosphate group of NADP⁺ resulting in an improvementin affinity and/or oxidation and/or reduction capacity of the diaphorasevariant compared to the wild-type diaphorase.

In one embodiment, the second electron transfer component of thecofactor regeneration system of the present invention comprises orconsists of a hydrogenase moiety, wherein said hydrogenase moietycomprises or consists of an amino acid sequence having at least 70%(such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%)sequence identity to the amino acid sequence of Ralstonia eutrophasoluble hydrogenase moiety (SEQ ID NOs: 24 and/or 25).

In one embodiment, the second electron transfer component of thecofactor regeneration system of the present invention comprises orconsists of a hydrogenase moiety, wherein said hydrogenase moietycomprises or consists of an amino acid sequence having at least 70%(such as at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%)sequence identity to the amino acid sequence of Ralstonia eutrophamembrane-bound hydrogenase moiety (SEQ ID NOs: 26 and/or 27 and/or 28).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Ralstonia eutropha regulatory hydrogenase moiety(SEQ ID NOs: 29, and/or 30).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Escherichia coli hydrogenase 1 (SEQ ID NOs:31and/or 32).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Escherichia coli hydrogenase 2 (SEQ ID NOs:33and/or 34).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Aquifex aeolicus hydrogenase 1 (SEQ ID NO:35and/or 36).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Hydrogenovibrio marinus hydrogenase (SEQ ID NOs:37 and/or 38).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Thiocapsa roseopersicina hydrogenase (SEQ ID NOs:39 and 40).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Alteromonas macleodii hydrogenase (SEQ ID NOs:41and/or 42).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Rhodococcus opacus soluble hydrogenase moiety(SEQ ID NOs: 43 and/or 44).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Allochromatium vinosum membrane bound hydrogenase(SEQ ID NOs: 45 and/or 46).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Desulfovibrio fructosovorans membrane boundhydrogenase (SEQ ID NOs: 47 and/20 or 48).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Clostridium pasteurianum iron-iron hydrogenase(SEQ ID NOs: 49).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Clostridium acetobutylicum iron-iron hydrogenase(SEQ ID NOs: 50).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Chlamydomonas reinhardtii iron-iron hydrogenase(SEQ ID NOs: 51).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Desulfomicrobium baculatum nickel-iron seleniumhydrogenase (SEQ ID NOs: 52 and/or 53).

In another embodiment, said hydrogenase moiety may comprise or consistof an amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Desulfovibrio vulgaris Nickel Iron hydrogenasepdb 1H₂A (SEQ ID NOs: 69 and/or 70).

In another embodiment, said hydrogenase moiety may comprise or consistof an 20 amino acid sequence having at least 70% (such as at least 75%,80%, 85%, 90%, 95%, 96%, 98%, 99% or 100%) sequence identity to theamino acid sequence of Desulfovibrio gigas Periplasmic [NiFe]hydrogenase (SEQ ID NOs: 71 and/or 72).

In one embodiment of the present invention, the second electron transfercomponent is a hydrogenase moiety, wherein said hydrogenase moiety doesnot comprise (or lacks) a flavin mononucleotide (FMN) prosthetic groupand/or a flavin adenine dinucleotide (FAD) prosthetic group. In oneembodiment of the present invention, the hydrogenase moiety which doesnot comprise (or lacks) a FMN group and/or FAD group has increasedstability compared to a hydrogenase comprising a FMN and/or a FAD group.Accordingly, use of such a hydrogenase (i.e. lacking a FMN and/or FADprosthetic group) as the second electron transfer component in acofactor regeneration system of the present invention may beadvantageous because it increases the robustness/stability of theoverall system. Examples of hydrogenases lacking a FMN prosthetic groupinclude Ralstonia eutropha membrane-bound hydrogenase (SEQ ID NOs:26-28), Ralstonia eutropha regulatory hydrogenase (SEQ ID NOs:29-30),Escherichia coli hydrogenase 1 (SEQ ID NOs:31-32), Escherichia colihydrogenase 2 (SEQ ID NOs:33-34), Aquifex aeolicus hydrogenase 1 (SEQ IDNOs:35-36), Hydrogenovibrio marinus membrane-bound hydrogenase (SEQ IDNOs: 37-38), Desulfovibrio vulgaris Nickel Iron hydrogenase (SEQ ID NOs:69-70) and Desulfovibrio gigas Periplasmic [NiFe] hydrogenase (SEQ IDNOs:71-72).

In one embodiment, the second electron transfer component comprises orconsists of non-biological nanoparticles. Suitable non-biologicalnanoparticles include metal nanoparticles (such as platinum or palladiumnanoparticles), or nanoparticles of a metal oxide, or nanoparticles of ametal sulphide (such as molybdenum disulfide). The non-biologicalnanoparticles of the present invention are able to catalyse theinterconversion of H⁺ and H₂ close to the thermodynamic potential of the2H⁺/H₂ couple under the experimental conditions. For example, the 2H⁺/H₂couple potential is −0.413 V at 25° C., pH 7.0 and 1 bar H₂. In oneembodiment, the second electron transfer component (i.e. thenon-biological nanoparticles and/or the hydrogenase) operates in H2oxidation at less than 100 mV more positive than the 2H⁺/H₂ couplepotential, and/or operates in H+ reduction at less than 100 mV morenegative than the 2H⁺/H₂ couple potential. Methods for determiningoverpotential are routine to those skilled in the art. Briefly, thecatalyst (hydrogenase or non-biological nanoparticle) is attached to anelectrode such that the catalyst can exchange electrons directly withthe electrode. The electrode is immersed in an electrochemical cellsolution containing buffered electrolyte (for example 50 mM phosphate atpH 7.0) saturated with H₂ (ie in equilibrium with a gas atmospherecomprising 100% H₂) and the electrode potential is cycled between lowerand upper limiting potentials, eg −0.6 V vs the standard hydrogenelectrode (SHE) and 0.2 V vs SHE. A catalyst which operates at minimaloverpotential shows an electrocatalytic H⁺ reduction current and/or anelectrocatalytic H₂ oxidation current close to (ie within 100 mV belowor above) the thermodynamic potential of the 2H⁺/H₂ couple under theexperimental conditions. For example, the 2H⁺/H₂ couple potential is−0.413 Vat 25° C., pH 7.0 and 1 bar H₂ or −0.36 V at 30° C., pH 6.0 and1 bar H₂. Reference: Vincent, K. A., Parkin, A., Lenz, O., Albracht, S.P. J., Fontecilla-Camps, J. C., Cammack, R., Friedrich, B., Armstrong,F. A., ‘Electrochemical Definitions of O₂ Sensitivity and OxidativeInactivation in Hydrogenases’ Journal of the American Chemical Society(2005) 127, 18179-18189.

In one embodiment of the present invention, the first and secondelectron transfer components do not occur together in nature as anenzyme complex. In other words, the first and second transfer componentsare not naturally associated with each other, and said first and secondelectron transfer components do not transfer electrons to and/or acceptelectrons from each other in the “natural” cellular environment. Firstand second electron transfer components of the present invention aretherefore selected from components which do not occur together in awild-type enzyme complex.

Thus, by way of example, the first and second electron transfercomponents may be selected from (or derived from) different bacterialspecies or from different bacterial genera. In another embodiment, thefirst and second electron transfer components may be selected from (orderived from) the same bacterial genus or species, but said first andsecond electron transfer components are selected from (or derived from)different enzymes. Taking Ralstonia eutropha as an example, the firstelectron transfer component may comprise the diaphorase HoxF and/orHoxU, and/or HoxH and/or HoxY and/or HoxI (SEQ ID NOs: 1 and/or 2, 24,25 and/or 3). In this scenario, the second electron transfer componentmay comprise any suitable hydrogenase moiety such as the membrane boundhydrogenase moiety HoxGKZ (SEQ ID NOs: 26-28), or the regulatoryhydrogenase moiety HoxBC (SEQ ID NOs: 29-30), but may not comprise thesoluble hydrogenase moiety HoxHY (SEQ ID NOs: 24-25), which is normallyassociated with the diaphorase HoxFU. Thus, the individual components ofthe cofactor regeneration system can be tuned (by choice of differentcomponents of the system) for a specific application and/orcondition(s). The Inventors have realized that by selecting componentswhich are not naturally associated with each other in nature allowsoptimization of the cofactor regeneration system depending on theapplication/conditions. For example, an oxygen (O₂)—tolerant hydrogenasemay be selected as the second electron transfer component if thecofactor regeneration system is used to supply cofactors to enzymesrequiring O₂, such as cytochrome P450 monoxygenases.

In another aspect the invention provides a cofactor regeneration systemcomprising or consisting of:

-   -   i) a first electron transfer component selected from a        polypeptide comprising a diaphorase variant as described herein,        and    -   ii) a second electron transfer component selected from a        hydrogenase moiety and/or non-biological nanoparticles, and    -   iii) an electronically conducting surface;        wherein the first and second electron transfer components are        immobilised on the electrically conducting surface.

Suitable second electron transfer components i.e. hydrogenase moietiesand non-biological particles are described above, and may be used in thepresent aspect of the invention.

As described herein, the cofactor regeneration system of the presentinvention may comprise an electronically conducting surface wherein thefirst and second electron transfer components are immobilised on theelectronically conducting surface. In one embodiment, the term“immobilised” embraces adsorption, entrapment and/or cross-linkage.Adsorption generally relies on a non-covalent interaction. Thus, in oneembodiment, the term “immobilised” refers to a non-covalent attachment.In another embodiment, the first and second electron transfer componentsare covalently bonded to the electronically conducting surface. Such aninteraction may be referred to as “cross-linked” attachment. Entrapmentrefers to the first and second electron transfer components beingtrapped within the electronically conducting surface (e.g. because theelectronically conducting surface is porous). Thus, in one embodiment,the term “immobilised” refers to entrapment. Entrapment may embracenon-covalent and/or covalent attachment. A combination of the abovementioned immobilisation means may also be used. In one embodiment, theelectronically conducting surface of the present invention serves totransfer electrons between said first and second electron transfercomponents of the cofactor regeneration system.

In one embodiment, the electronically conducting surface of the cofactorregeneration system comprises or consists a carbon material, such asgraphite particles, carbon nanotubes, carbon black, activated carbon,carbon nanopowder vitreous carbon (glassy carbon), carbon fibres, carboncloth, carbon felt, carbon paper, graphene, glassy carbon, highlyordered pyrolytic graphite or edge oriented pyrolytic graphite;

Other suitable electronically conducting surface materials may compriseor consist of gold, silver, tungsten, iridium, metal oxide nanoparticlessuch as titanium oxide, indium oxide, tin oxide, indium tin oxide, andmetal sulphide nanoparticles such as (boron) doped diamond.Electronically conducting surfaces combining one or more of theabove-mentioned materials are also embraced by the present invention.

In one embodiment of the present invention, the cofactor regenerationsystem advantageously comprises or consists of particles (e.g. beads)which can easily be removed from a reaction mixture, for example bysedimentation, filtration and/or centrifugation. The cofactorregeneration system comprising said particles can thereforeadvantageously be readily separated from a reaction mixture (e.g. bysedimentation, filtration and/or centrifugation) and re-used. In anotherembodiment, if the electronically conducting surface utilised is carboncloth, carbon felt, carbon paper, (or similar material) for example,then the cofactor regeneration system can be readily separated from areaction mixture by simply removing said carbon cloth/felt or paper fromsaid reaction mixture.

In one embodiment, the term ‘electronically conducting surface’ of thepresent invention embraces one or more electronically conductingsurfaces (e.g. a carbon material as described herein) connected/coupledvia an electronically conducting linker (e.g. a wire). By way ofexample, two carbon particles may be connected via said electronicallyconducting linker, with the first and second electron transfercomponents of the present invention immobilised on said carbonparticles. The electronically conducting linker may be made from one ofthe non-carbon materials described herein (e.g. gold, silver, tungsten,iridium etc) or another metal such as copper or aluminium. In oneembodiment, the first electron transfer component of the presentinvention is immobilised to an electronically conducting carbon surfaceand the second electron transfer component is attached to a secondelectronically conducting carbon surface, wherein said carbon surfacesare connected via an electronically conducting linker. In other words,the electronically conducting surface of the present invention embracesone or more electronically conducting surfaces (e.g. a carbon materialas described herein) coupled/connected/inter-linked by an electronicallyconducting linker (e.g. a wire made of gold, silver, tungsten, iridium)or another metal such as copper or aluminium.

In one embodiment, the cofactor regeneration system of the presentinvention further comprises or consists of one or more cofactorsselected from NAD⁺, NADH, NADP⁺ and/or NADPH.

In use, electrons flow between the first and second electron transfercomponents of the cofactor regeneration system of the present invention.In one embodiment, electrons flow from the first electron transfercomponent to the second transfer component. Alternatively, electrons mayflow from the second transfer component to the first electron transfercomponent. In use, the flow of electrons may be reversed, depending onreaction conditions. In use, electrons may flow directly between thefirst and second electron transfer components or indirectly via theelectronically conducting surface of the present invention.

In one embodiment, the cofactor regeneration system of the presentinvention further comprises an oxidoreductase selected from adehydrogenase, reductase, oxidase, synthase, transhydrogenase,dioxygenase, mono-oxygenase cytochrome p450 monooxygenase and/or enereductase. Variants, derivatives and functional fragments of theaforementioned oxidoreductases are also embraced by the presentinvention. In one embodiment, the cofactor regeneration system of thepresent invention further comprises a variant of the aforementionedoxidoreductases, wherein said variant retains at least some of theactivity/functionality of the native/wild-type enzyme. In oneembodiment, the variant oxidoreductase has increased/improvedactivity/functionality when compared to the native/wild-type enzyme.

In one embodiment said oxidoreductase selected from a dehydrogenase,reductase, oxidase, synthase, transhydrogenase, dioxygenase,mono-oxygenase, cytochrome p450 monooxygenase and/or ene reductase isimmobilised on said electronically conducting surface. Suitableimmobilisation methods are described above. Alternatively, theaforementioned oxidoreductases may be immobilised on a separate/distinctelectronically conducting surface from the first and second electrontransfer components of the cofactor regeneration system. In anotherembodiment, said oxidoreductases selected from a dehydrogenase,reductase, oxidase, synthase, transhydrogenase, dioxygenase,mono-oxygenase, cytochrome p450 monooxygenase and/or ene reductase arenot immobilised, but are present in a solution containing the cofactorregeneration system of the present invention. This approach would beparticularly suited to those proteins/protein complexes which cannot bereadily be immobilised. By way of example, multi-redox componentcytochrome P450 mono-oxygenases or dioxygenases may be provided in asolution with the cofactor regeneration system of the present invention.

In one aspect, the present invention provides a cofactor regenerationsystem and components thereof (as defined above) for use in a method forgenerating a cofactor.

In another aspect, the present invention provides a cofactorregeneration system and components thereof (as defined above) for use ina method for regenerating a cofactor.

Thus, in one aspect the invention provides a method for (re)generating acofactor comprising or consisting of adding to the cofactor regenerationsystem described herein, a cofactor selected from NAD⁺, NADH, NADP⁺and/or NADPH.

In one embodiment, the NADH, NAD, NADPH or NADP added is present at aconcentration of 1 μM to 1M (such as 1 μM to 800 mM, 1 μM to 600 mM, 1μM to 400 mM, 1 μM to 200 mM, 1 μM to 100 mM, 1 μM to 10 mM, or 1 μM to1 mM). In another embodiment, the NADH, NAD, NADPH or NADP added ispresent at a concentration of 1 μM to 10 mM (such as 5 μM to 10 mM, 10μM to 10 mM, 25 μM to 10 mM, 50 μM to 10 mM, 100 μM to 10 mM, 250 μM to10 mM, 500 μM to 10 mM or 1 mM to 10 mM). In one embodiment, the NADH,NAD, NADPH or NADP added is present at a concentration of 0.2 mM or 1-2mM.

In one embodiment, the invention provides a method for (re)generatingNAD⁺ comprising or consisting of: adding to the cofactor regenerationsystem described herein, NADH and a gas atmosphere comprising an inertgas. The NADH added may be present at a concentration of 1 μM to 1M(such as 1 μM to 800 mM, 1 μM to 600 mM, 1 μM to 400 mM, 1 μM to 200 mM,1 μM to 100 mM, 1 μM to 10 mM, or 1 μM to 1 mM) or 1 μM to 10 mM (suchas 5 μM to 10 mM, 10 μM to 10 mM, 25 μM to 10 mM, 50 μM to 10 mM, 100 μMto 10 mM, 250 μM to 10 mM, 500 μM to 10 mM or 1 mM to 10 mM). The inertgas may be selected, for example, from N₂ or Argon (Ar). In oneembodiment the inert gas is present at a concentration of 80-100% (i.e.the gas is present in the headspace of a suitable container comprisingthe cofactor regeneration system)

In another embodiment, the invention provides a method for(re)generating NADH comprising or consisting of: adding to the cofactorregeneration system described herein, NAD⁺ and a gas atmospherecomprising H2 and O2. The NAD⁺ added may be present at a concentrationof 1 μM to 1M (such as 1 μM to 800 mM, 1 μM to 600 mM, 1 μM to 400 mM, 1μM to 200 mM, 1 μM to 100 mM, 1 μM to 10 mM, or 1 μM to 1 mM) or 1 μM to10 mM (such as 5 μM to 10 mM, 10 μM to 10 mM, 25 μM to 10 mM, 50 μM to10 mM, 100 μM to 10 mM, 250 μM to 10 mM, 500 μM to 10 mM or 1 mM to 10mM). In one embodiment, the H₂ is present at a concentration of 1-100%,with the remaining gas comprising an inert gas. In one embodiment, theH₂ is present at a concentration of 80-100% and the O₂ is present at aconcentration of 0-20% (i.e. the gas is present in the headspace of asuitable container comprising the cofactor regeneration system.). Inanother embodiment, the H₂ is present at a concentration of 1-4% and theO₂ is present at a concentration of 96-99% (i.e. the gas is present inthe headspace of a suitable container comprising the cofactorregeneration system). In one embodiment, the H₂ is present at aconcentration of 1-4% in air, or at a concentration of 70-99% in air.

In another embodiment, the invention provides a method for(re)generating NADP comprising or consisting of: adding to the cofactorregeneration system described herein, NADPH and a gas atmospherecomprising an inert gas. The NADPH added may be present at aconcentration of 1 μM to 1M (such as 1 μM to 800 mM, 1 μM to 600 mM, 1μM to 400 mM, 1 μM to 200 mM, 1 μM to 100 mM, 1 μM to 10 mM, or 1 μM to1 mM) or 1 μM to 10 mM (such as 5 μM to 10 mM, 10 μM to 10 mM, 25 μM to10 mM, 50 μM to 10 mM, 100 μM to 10 mM, 250 μM to 10 mM, 500 μM to 10 mMor 1 mM to 10 mM). The inert gas may be selected, for example, from N₂or Argon (Ar). In one embodiment the inert gas is present at aconcentration of 80-100% (i.e. the gas is present in the headspace of asuitable container comprising the cofactor regeneration system).

In another embodiment, the invention provides a method for(re)generating NADPH comprising or consisting of: adding to the cofactorregeneration system described herein, NADP⁺ and a gas atmospherecomprising H₂. The NADP⁺ added may be present at a concentration of 1 μMto 1M (such as 1 μM to 800 mM, 1 μM to 600 mM, 1 μM to 400 mM, 1 μM to200 mM, 1 μM to 100 mM, 1 μM to 10 mM, or 1 μM to 1 mM) or 1 μM to 10 mM(such as 5 μM to 10 mM, 10 μM to 10 mM, 25 μM to 10 mM, 50 μM to 10 mM,100 μM to 10 mM, 250 μM to 10 mM, 500 μM to 10 mM or 1 mM to 10 mM).).In one embodiment, the H₂ is present at a concentration of 1-100% (i.e.the gas is present in the headspace of a suitable container comprisingthe cofactor regeneration system). In one embodiment, the H₂ is presentat a concentration of 80-100%. In one embodiment, the H₂ is present atless than 3%, such as 2% or 1%.

Other inert gases suitable for use in the methods of the presentinvention include Helium (He), Neon (Ne), Krypton (Kr), Xenon (Xe),Radon (Rn) and/or Sulfur hexafluoride (SF₆),In one embodiment, the gasphase in contact with the cofactor regeneration system of the presentinvention comprises or consists of one or more gases that are notconsidered inert gases. Examples of such ‘non-inert’ gases includeammonia (NH₃), carbon dioxide (CO₂), and/or hydrogen sulphide (H₂S).

In one embodiment, the methods described herein further comprises orconsists of harvesting the (re)generated cofactor by removing thecofactor regeneration particles by filtration or by centrifugation, orby allowing the particles to settle and decanting off the solution (i.e.sedimentation). Filtration methods are known to those skilled in theart, and any such method may be used. By way of example, a simple filterpaper may be used to remove the cofactor regeneration particles.

In another aspect, the invention provides use of a cofactor regenerationsystem as described herein, or a diaphorase variant for use in acofactor regeneration system of the present invention, for generating acofactor.

In another aspect, the invention provides use of a cofactor regenerationsystem as described herein, or a diaphorase variant for use in acofactor regeneration system of the present invention, for regeneratinga cofactor.

In one embodiment, the invention provides use of a cofactor regenerationsystem or use of a diaphorase variant, as described herein, in asynthetic reaction. In one embodiment the synthetic reaction is anenzyme-catalyzed synthetic reaction. Suitable applications for thecofactor regeneration system of the present invention include syntheticprocesses wherein cofactor-dependent oxidoreductases are used ascatalysts.

In another aspect, the invention provides a synthetic reactioncomprising or consisting of the cofactor regeneration system asdescribed herein, or the diaphorase variant as described herein.

In one embodiment, the synthetic reaction further comprises anoxidoreductase which is dependent on NAD+, NADH, NADP+ and/or NADPH.Examples of oxidoreductases include dehydrogenase, reductase, oxidase,synthase, transhydrogenase, dioxygenase, mono-oxygenase, cytochrome p450monooxygenase and/or ene reductase.

In one embodiment, said oxidoreductase is immobilised on theelectronically conducting surface of the present invention along withthe first and second electron transfer components of the cofactorregeneration system of the present invention. Suitable methods forimmobilisation have been described elsewhere in the presentspecification and apply equally hereto. Thus, immobilisation embracescovalent and non-covalent attachments including adsorption, entrapmentand/or cross-linkage. Alternatively, the aforementioned oxidoreductasesmay be immobilised on a separate/distinct electronically conductingsurface from the first and second electron transfer components of thecofactor regeneration system. In another embodiment, saidoxidoreductases selected from a dehydrogenase, reductase, oxidase,synthase, transhydrogenase, dioxygenase, mono-oxygenase, cytochrome p450monooxygenase and/or ene reductase are not immobilised, but are presentin a solution containing the cofactor regeneration system of the presentinvention. This approach would be particularly suited to thoseproteins/protein complexes which cannot be readily be immobilised. Byway of example, multi-redox component cytochrome P450 mono-oxygenases ordioxygenases may be provided in a solution with the cofactorregeneration system of the present invention

In one embodiment, the cofactor regeneration system of the presentinvention is employed to supply NADH to an NADH-dependent oxidoreductase(such as a NADH-dependent dehydrogenase) which is immobilised on theelectronically conducting surface of the present invention (i.e. inaddition to the first and second electron transfer components of thepresent invention).

In one embodiment, the cofactor regeneration system of the presentinvention is employed to supply NADH to an NADH-dependant oxidoreductaserequiring O₂ (such as a NADH-dependent cytochrome P450 mono-oxygenase)which is immobilised on the electronically conducting surface of thepresent invention (i.e. in addition to the first and second electrontransfer components of the present invention).

In one embodiment, the cofactor regeneration system of the presentinvention is employed to supply of NAD⁺ to an NAD⁺-dependantoxidoreductase (such as a NAD⁺-dependent dehydrogenase) which isimmobilised on the electronically conducting surface of the presentinvention (i.e. in addition to the first and second electron transfercomponents of the present invention).

In one embodiment, the cofactor regeneration system of the presentinvention is employed to supply of NADP⁺ to an NADP⁺-dependantoxidoreductase (such as a NADP⁺-dependent aldehyde dehydrogenase) whichis immobilised on the electronically conducting surface of the presentinvention (i.e. in addition to the first and second electron transfercomponents of the present invention).

In one embodiment, the cofactor regeneration system of the presentinvention is employed to supply of NADPH to an NADPH-dependentoxidoreductase (such as a NADPH-dependent carbonyl reductase or aNADPH-dependent cytochrome P450 mono-oxygenase) which is immobilised onthe electronically conducting surface of the present invention (i.e. inaddition to the first and second electron transfer components of thepresent invention).

In a further aspect, the invention provides a kit comprising orconsisting of the cofactor regeneration system as described herein, orthe diaphorase variant as described herein, and a cofactor selected fromNAD⁺, NADH, NADP⁺ and/or NADPH. In one embodiment, the kit furthercomprises an oxidoreductase such as a dehydrogenase, a mono-oxygenaseand/or a cytochrome p450 monooxygenase.

In another aspect of the present invention, there is provided a DNAsequence that encodes the first and/or second electron transfercomponents of the cofactor regeneration system of the present invention.In one embodiment, the present invention provides a DNA sequence of anyof the SEQ ID NOs (i.e. any of SEQ ID NOs 1-72) representing the firstand second electron transfer components described herein. In oneembodiment, the DNA sequence is prepared as part of a DNA vector,wherein the vector comprises a promoter and terminator. In oneembodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lacoperator IPTG 0.2 mM (0.05-2.0 mM) AcoE Acetoin 0.15% (0.05-0.5%) SHpromoter self inducing during growth in Fructose-Glycerol-minimal medium

The DNA construct of the present invention may be designed in silico,and then synthesised by conventional DNA synthesis techniques.

The above-mentioned DNA sequence information is optionally modified forcodon-biasing according to the ultimate host cell (e.g. E. coli)expression system that is to be employed.

Methods for expression of proteins in cellular (e.g. microbial)expression systems are well known and routine to those skilled in theart. In one embodiment, the first and second electron transfercomponents of the present inventions are encoded and expressed from thesame vector in an appropriate host cell (e.g. a microbial cell, such asE. coli). In another embodiment, the first and second electron transfercomponents of the present invention are encoded and expressed fromdifferent/separate vectors in the same or different host cells. Theextraction of the first and electron transfer components from said hostcells post-expression can be achieved through routine methods known tothose skilled in the art.

In one embodiment, the first and second electron transfer components ofthe present invention are expressed in the same host cell (e.g. amicrobial host cell, such as E. coli) as an oxidoreductase selected froma dehydrogenase, reductase, oxidase, synthase, transhydrogenase,dioxygenase, mono-oxygenase, cytochrome p450 monooxygenase and/or enereductase. Said oxidoreductase may be expressed from the same and/or adifferent/separate vector from the first and/or second electron transfercomponents.

Reeve, H. A., Lauterbach, L., Ash, P. A., Lenz, O., Vincent, K. A., ‘Amodular system for regeneration of NAD cofactors using graphiteparticles modified with hydrogenase and diaphorase moieties’ Chem.Commun. 2012, 48 (10), 1589-1591 is incorporated herein, in itsentirety, by reference thereto.

Definitions

Sequence Homology:

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art. Global methods align sequences fromthe beginning to the end of the molecule and determine the bestalignment by adding up scores of individual residue pairs and byimposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W,see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving theSensitivity of Progressive Multiple Sequence Alignment Through SequenceWeighting, Position—Specific Gap Penalties and Weight Matrix Choice,22(22) Nucleic Acids Research 4673-4680 (1994); and iterativerefinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracyof Multiple Protein. Sequence Alignments by Iterative Refinement asAssessed by Reference to Structural Alignments, 264(4) J. Mol. Biol.823-838 (1996). Local methods align sequences by identifying one or moreconserved motifs shared by all of the input sequences. Non-limitingmethods include, e.g., Match-box, see, e.g., Eric Depiereux and ErnestFeytmans, Match-Box: A Fundamentally New Algorithm for the SimultaneousAlignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992);Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting SubtleSequence Signals: A Gibbs Sampling Strategy for Multiple Alignment,262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle etal., Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics:1428-1435 (2004). Thus, percent sequenceidentity is determined by conventional methods. See, for example,Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, twoamino acid sequences are aligned to optimize the alignment scores usinga gap opening penalty of 10, a gap extension penalty of 1, and the“blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shownbelow (amino acids are indicated by the standard one-letter codes).

Alignment Scores for Determining Sequence Identity

A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2 −2 1 6C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1−2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2−3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3−1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2−2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3−3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

The percent identity is then calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{bmatrix}{{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}\mspace{14mu}{number}} \\{{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}} \\{{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}}\end{bmatrix}} \times 100$

“R” is the standard nomenclature for a degeneracy of A or G at thisposition in a nucleotide sequence.

The present invention will now be described, by way of example only,with reference to the accompanying Examples and Figures, in which:

FIG. 1 shows UV-visible spectra demonstrating generation of NADH from asolution of NAD⁺ (1 mM) saturated with H₂ by a cofactor regenerationsystem of the present invention consisting of pyrolytic graphiteparticles modified with Ralstonia eutropha soluble hydrogenasediaphorase domain (component (i)), and Escherichia coli hydrogenase 2(component (ii)). The solution also contained potassium phosphatebuffer, 50 mM, pH 7.0. The reaction was carried out at 20° C. Thecharacteristic peak at 340 nm indicates NADH formation. Solid line:before addition of H₂; dashed line: 4.5 hours after addition of H₂.

FIG. 2 shows a UV-visible spectrum demonstrating generation of NADH froma solution of NAD⁺ (1 mM) saturated with a gas mixture of 99% H₂: 1% O₂by a cofactor regeneration system of the present invention consisting ofpyrolytic graphite particles modified with Ralstonia eutropha solublehydrogenase HoxFU diaphorase domain (component (i)), and Ralstoniaeutropha membrane-bound hydrogenase (component (ii)). The solution alsocontained potassium phosphate buffer, 50 mM, pH 7.0. The reaction wascarried out at 20° C. The characteristic peak at 340 nm indicates NADHformation.

FIG. 3 shows a UV-visible spectrum demonstrating generation of NADH froma solution of NAD⁺ (2 mM) by a cofactor regeneration system of thepresent invention consisting of pyrolytic graphite particles modifiedwith Ralstonia eutropha HoxFU (component (i)) and Escherichia colihydrogenase-1 (component (ii)). A gas mixture of 1% O₂ and 99% H₂ wascontinually flowed through the head space of the reaction vial. Thesolution also contained bis-Tris buffer, 50 mM, pH 6. The reaction wascarried out at 20° C. The peak at 340 nm indicates NADH formation.

FIG. 4 shows a plot of NADH concentration over time during generation ofNAD⁺ from a solution of NADH (1 mM) saturated with N₂ by a cofactorregeneration system of the present invention consisting of pyrolyticgraphite particles modified with Escherichia coli hydrogenase 2(component (ii)) and Ralstonia eutropha H₁₆ soluble hydrogenasediaphorase domain (HoxFU, component (i)). The solution also containedpotassium phosphate buffer, 50 mM, pH 7.0. The reaction was carried outat 20° C. Samples were removed for analysis at the time pointsindicated. The concentration of NADH was determined by comparison of theratio of absorbance at 260 nm to absorbance at 340 nm with the ratio forsamples of known concentration ratio of NAD to NADH.

FIG. 5 shows UV-visible spectra demonstrating generation of NADH from asolution of NAD⁺ (0.8 mM) saturated with H₂ by a cofactor regenerationsystem of the present invention consisting of pyrolytic graphiteparticles first modified with platinum (component (ii)), and then withRalstonia eutropha soluble hydrogenase diaphorase domain (component(i)). The solution also contained potassium phosphate buffer, 50 mM, pH7.0. The reaction was carried out at 20° C. The characteristic peak at340 nm indicates NADH formation.

FIG. 6 shows a cyclic voltammogram demonstrating the response of anunmodified electrode (upper two lines) and an electrode modified withthe HoxFUHY tetramer of Ralstonia eutropha soluble hydrogenase bearing asite directed mutation in the NAD⁺ binding pocket which was intended toincrease affinity for NADP⁺ (lower two lines). Reduction of NADP⁺ atminimal overpotential demonstrates the capacity for regenerating NADPHwith electrons from H₂ using the cofactor regeneration system of thepresent invention with a variant of Ralstonia eutropha HoxFU diaphorasedimer in place of the native diaphorase dimer. The electrode ispyrolytic graphite ‘edge’, and is immersed in a solution containing 2 mMNADP⁺ in Tris-HCl buffer, pH 8.0, at 30° C.

FIG. 7 shows a schematic of NADH regeneration using electrons from H₂ bypyrolytic graphite (PG) particles modified with a diaphorase (HoxFUsubunits of Ralstonia eutropha soluble hydrogenase, component (i)) and ahydrogenase or Pt (component (ii)); ▪ represents an iron sulfur electronrelay cluster; * represents a catalytic active site. By appropriatechoice of enzymes and conditions, the direction of catalysis can bereversed. In this schematic, NADH is supplied to an NADH-dependentoxidoreductase to support the transformation of substrate to product.

FIG. 8 shows that cofactor regeneration particles of the presentinvention can supply NADH to a cofactor dependent dehydrogenase.Attentuated Total Reflectance (ATR)-Infrared (IR) spectra of thesupernatant solution before (dashed) and 7 hours after (solid)initiation by H₂ show conversion of pyruvate to lactate by lactatedehydrogenase (0.5 mg/ml). The dehydrogenase was supplied only withpyruvate (3 mM), NAD⁺ (0.2 mM) and pyrolytic graphite particles modifiedwith HoxFU and E. coli Hydrogenase-2 in pH 7.0, 50 mM potassiumphosphate buffer. ATR-IR spectra were recorded using a Bio-Rad FTS-6000FTIR spectrometer equipped with a diamond attenuated total reflectanceaccessory (DurasampIIR II, SensIR Technologies). The peak at 1175 cm⁻¹demonstrates pyruvate consumption. The peaks at 1120 cm⁻¹ and 1040 cm⁻¹demonstrate lactate formation.

FIG. 9 shows a plot of NADH concentration over time during generation ofNADH from a solution of NAD⁺ (1 mM) by a cofactor regeneration system ofthe present invention consisting of pyrolytic graphite particlesmodified with Ralstonia eutropha soluble hydrogenase diaphorase domainHoxFU (component (i)), and Escherichia coli hydrogenase 2 (component(ii)). o represents a data series from an experiment performed under agas mixture of 99% N₂: 1% H₂ at 1 bar. ▪ represents a data series froman experiment performed under 100% H₂ at 1 bar. The solution alsocontained potassium phosphate buffer, 50 mM, pH 7.0. The reaction wascarried out at 20° C. Aliquots were taken at the times indicated; aratio of the UV-Vis spectra peaks at 260 nm and 340 nm was used tocalculate the concentration of NADH by comparison with a standard curve.100% conversion of NAD⁺ to NADH is achieved after 60 minutes at 100% H₂.

FIG. 10 shows a UV-visible spectrum demonstrating generation of NADHfrom a solution of NAD⁺ (0.8 mM) saturated with H₂ at 1 bar by acofactor regeneration system of the present invention consisting ofpyrolytic graphite particles modified with soluble extract of Ralstoniaeutropha HoxHYFU 164A variant in which the hydrogenase activity of HoxHYsubunits is inactivated (component (i)), and Desulfovibrio vulgarisMiyazaki F Nickel-Iron hydrogenase (component (ii)). The solution alsocontained bis-Tris buffer, 100 mM, pH 6.0. The reaction was carried outat 20° C. The characteristic peak at 340 nm indicates NADH formation.

FIG. 11 shows UV-visible spectra demonstrating generation of NADH from asolution of NAD⁺ (2 mM) saturated with H₂ at 1 bar by a cofactorregeneration system of the present invention consisting of carbon paper(solid line); carbon nanotubes (dotted line) or carbon nanopowder(dashed line) modified with Ralstonia eutropha HoxFU (component (i)) andDesulfovibrio vulgaris Miyazaki F hydrogenase (component (ii)). Thesolution also contained mixed buffer, 100 mM, pH 7.0. The reaction wascarried out at 20° C. The characteristic peak at 340 nm indicates NADHformation.

FIG. 12 shows a UV-visible spectrum demonstrating generation of NADPHfrom a solution of NADP⁺ (2 mM) saturated with H₂ at 1 bar by a cofactorregeneration system of the present invention consisting of pyrolyticgraphite particles modified with a variant of Ralstonia eutropha solublehydrogenase (D467S E341A) (component (i)), and Desulfovibrio vulgarisMiyazaki F hydrogenase (component (ii)). The solution also containedbis-Tris buffer, 100 mM, pH 6.0. The reaction was carried out at 20° C.The characteristic peak at 340 nm indicates NADPH formation.

FIG. 13A shows a gas chromatogram of heptanal and heptanol standards.Heptanol and heptanal were extracted with ethyl acetate (300 μL) from anaqueous solution containing heptanol (1 mM), heptanal (1 mM) in pH 6,100 mM bis-Tris buffer (1 mL).

FIG. 13B shows that cofactor regeneration particles of the presentinvention can supply NADH to a cofactor dependent dehydrogenase.Pyrolytic graphite particles were modified with soluble extract ofRalstonia eutropha soluble hydrogenase 164A variant (component (i)) andDesulfovibrio vulgaris Miyazaki F hydrogenase (component (ii)).Saccharomyces cerevisiae (yeast) alcohol dehydrogenase was included inthe reaction solution (1500 Units) and was supplied with heptanal (10mM) and NAD⁺ (2 mM). The solution also contained bis-Tris buffer, 100mM, pH 6.0. The reaction was carried out at 20° C. under a H₂ atmosphereat 1 bar. At the end of the experiment the heptanal and heptanol wereextracted using ethyl acetate (300 μL). The gas chromatogram wascollected on a ThermoFinnigan Trace GC. Comparison with the chromatogramof panel (a) confirms conversion of heptanal to heptanol.

KEY TO SEQ ID NOS

SEQ ID NO: 1 Ralstonia eutropha soluble hydrogenase diaphorase HoxF

SEQ ID NO: 2 Ralstonia eutropha soluble hydrogenase diaphorase HoxU

SEQ ID NO: 3 Ralstonia eutropha soluble hydrogenase diaphorase HoxI

SEQ ID NO: 4 flavoprotein (Fp) subcomplex of Bos taurus Complex 1, 51kDa

SEQ ID NO: 5 flavoprotein (Fp) subcomplex of Bos taurus Complex 1, 24kDa

SEQ ID NO: 6 Ralstonia eutropha NAD⁺-dependent formate dehydrogenase,diaphorase moiety (FdsB)

SEQ ID NO: 7 Ralstonia eutropha NAD⁺-dependent formate dehydrogenase,diaphorase moiety (FdsG)

SEQ ID NO: 8 NADPH oxidoreductase moiety from Pyrococcus furiosussoluble hydrogenase I gamma subunit

SEQ ID NO: 9 NADPH oxidoreductase moiety from Pyrococcus furiosussoluble hydrogenase I beta subunit

SEQ ID NO: 10 NADPH oxidoreductase moiety from Pyrococcus furiosussoluble hydrogenase II gamma subunit

SEQ ID NO: 11 NADPH oxidoreductase moiety from Pyrococcus furiosussoluble hydrogenase II beta subunit

SEQ ID NO: 12 Diaphorase moiety of Rhodococcus opacus SH HoxF

SEQ ID NO: 13 Diaphorase moiety of Rhodococcus opacus SH HoxU

SEQ ID NO: 14 Diaphorase moiety of Allochromatium vinosum SH, HoxF

SEQ ID NO: 15 Diaphorase moiety of Allochromatium vinosum SH, HoxU

SEQ ID NO: 16 Diaphorase moiety of Thiocapsa roseopersicina Hox1F

SEQ ID NO: 17 Diaphorase moiety of Thiocapsa roseopersicina Hox1U

SEQ ID NO: 18 Diaphorase moiety of Thiocapsa roseopersicina Hox2F

SEQ ID NO: 19 Diaphorase moiety of Thiocapsa roseopersicina Hox2U

SEQ ID NO: 20 Diaphorase moiety of Synechocystis sp. PCC 6803 HoxF

SEQ ID NO: 21 Diaphorase moiety of Synechocystis sp. PCC 6803 HoxU

SEQ ID NO: 22 Diaphorase moiety of Synechococcus elongatus PCC 6301 HoxF

SEQ ID NO: 23 Diaphorase moiety of Synechococcus elongatus PCC 6301 HoxU

SEQ ID NO: 24 Ralstonia eutropha soluble hydrogenase moiety (HoxH)

SEQ ID NO: 25 Ralstonia eutropha soluble hydrogenase moiety (HoxY)

SEQ ID NO: 26 Ralstonia eutropha membrane-bound hydrogenase moiety(HoxG)

SEQ ID NO: 27 Ralstonia eutropha membrane-bound hydrogenase moiety(HoxK)

SEQ ID NO: 28 Ralstonia eutropha membrane-bound hydrogenase moiety(HoxZ)

SEQ ID NO: 29 Ralstonia eutropha regulatory hydrogenase moiety (HoxB)

SEQ ID NO: 30 Ralstonia eutropha regulatory hydrogenase moiety (HoxC)

SEQ ID NO: 31 Escherichia coli hydrogenase 1 (large subunit)

SEQ ID NO: 32 Escherichia coli hydrogenase 1 (small subunit)

SEQ ID NO: 33 Escherichia coli hydrogenase 2 (large subunit)

SEQ ID NO: 34 Escherichia coli hydrogenase 2 (small subunit)

SEQ ID NO: 35 Aquifex aeolicus hydrogenase 1 (large subunit)

SEQ ID NO: 36 Aquifex aeolicus hydrogenase 1 (small subunit)

SEQ ID NO: 37 Hydrogenovibrio marinus hydrogenase (large subunit)

SEQ ID NO: 38 Hydrogenovibrio marinus hydrogenase (small subunit)

SEQ ID NO: 39 Thiocapsa roseopersicina hydrogenase HupL

SEQ ID NO: 40 Thiocapsa roseopersicina hydrogenase HupS

SEQ ID NO: 41 Alteromonas macleodii hydrogenase small subunit

SEQ ID NO: 42 Alteromonas macleodii hydrogenase large subunit

SEQ ID NO: 43 Rhodococcus opacus SH hydrogenase moiety HoxH

SEQ ID NO: 44 Rhodococcus opacus SH hydrogenase moiety HoxY

SEQ ID NO: 45 Allochromatium vinosum Membrane Bound Hydrogenase largesubunit

SEQ ID NO: 46 Allochromatium vinosum Membrane Bound Hydrogenase smallsubunit

SEQ ID NO: 47 Desulfovibrio fructosovorans nickel-iron hydrogenase largesubunit

SEQ ID NO: 48 Desulfovibrio fructosovorans nickel-iron hydrogenase smallsubunit

SEQ ID NO: 49 Clostridium pasteurianum iron-iron hydrogenase 1

SEQ ID NO: 50 Clostridium acetobutylicum iron-iron hydrogenase

SEQ ID NO: 51 Chlamydomonas reinhardtii iron-iron hydrogenase

SEQ ID NO: 52 Desulfomicrobium baculatum nickel-iron-seleniumhydrogenase large subunit

SEQ ID NO: 53 Desulfomicrobium baculatum nickel-iron-seleniumhydrogenase small subunit

SEQ ID NO: 54 Ralstonia eutropha diaphorase HoxF SH variant D326K(strain SH₁₃₄₄)

SEQ ID NO: 55 Ralstonia eutropha diaphorase HoxF SH variant D401K,strain SH₁₃₇₀

SEQ ID NO: 56 Ralstonia eutropha diaphorase HoxF SH variant D467S,strain SH₁₃₈₃

SEQ ID NO: 57 Ralstonia eutropha diaphorase HoxF SH variant D340A,strain SH₈₄₁

SEQ ID NO: 58 Ralstonia eutropha diaphorase HoxF SH variant E341A,strain SH₈₂₁

SEQ ID NO: 59 Ralstonia eutropha diaphorase HoxF SH variant SHD340A/E341A

SEQ ID NO: 60 Ralstonia eutropha diaphorase HoxF SH variant D340A/D401K

SEQ ID NO: 61 Ralstonia eutropha diaphorase HoxF SH variant D326K D401K

SEQ ID NO: 62 Ralstonia eutropha diaphorase HoxF SH variant D467S D401K

SEQ ID NO: 63 Ralstonia eutropha diaphorase HoxF SH variant D340N D467S

SEQ ID NO: 64 Ralstonia eutropha diaphorase HoxF SH variant E341A D467S

SEQ ID NO: 65 Ralstonia eutropha diaphorase HoxF SH variant E341H

SEQ ID NO: 66 Rhodobacter capsulatus diaphorase moiety of formateNAD⁺-reducing formate dehydrogenase from SB 1003, beta subunit FdsB

SEQ ID NO: 67 Rhodobacter capsulatus diaphorase moiety of formateNAD⁺-reducing formate dehydrogenase SB 1003, gamma subunit FdsG:

SEQ ID NO: 68 Ralstonia eutropha soluble hydrogenase moiety variantHoxH_I64A

SEQ ID NO: 69 Desulfovibrio vulgaris Nickel Iron hydrogenase Smallsubunit, pdb 1H₂A, Chain S

SEQ ID NO: 70 Desulfovibrio vulgaris Nickel Iron hydrogenase Largesubunit, pdb 1H₂A, Chain L

SEQ ID NO: 71 Desulfovibrio gigas Periplasmic [NiFe] hydrogenase Smallsubunit

SEQ ID NO: 72 Desulfovibrio gigas Periplasmic [NiFe] hydrogenase Largesubunit

SEQ ID NO: 73 Ralstonia eutropha hoxF nucleotide sequence

SEQ ID NO: 74 Ralstonia eutropha hoxU nucleotide sequence

SEQ ID NO: 75 Ralstonia eutropha hoxK nucleotide sequence

SEQ ID NO: 76 Ralstonia eutropha hoxG nucleotide sequence

SEQ ID NO: 77 Ralstonia eutropha hoxZ nucleotide sequence

EXAMPLES Example 1 NADH Cofactor Regeneration System

All steps were carried out in an anaerobic glove box (Glove BoxTechnology or MBraun) under an atmosphere of N₂. Particles of pyrolyticgraphite were prepared by abrasion of a piece of pyrolytic graphite withemery paper. These particles were immersed in 50 mM potassium phosphatebuffer and suspended by sonication (5 minutes, ultrasonic bath). Analiquot of the particle suspension was removed. To this aliquot wasadded an aliquot of Escherichia coli hydrogenase 2 (component (ii),10⁻¹² moles) and an aliquot of Ralstonia eutropha diaphorase (component(i), HoxFU, 10⁻¹¹ moles). The particles and enzymes were left at 4° C.for 10 minutes to allow the enzyme components to adsorb onto theparticles. Centrifugation (5 minutes, benchtop centrifuge) was used toseparate the particles and to remove excess unadsorbed enzyme. Theenzyme-modified particles were then resuspended in 1 mM NAD⁺ inpotassium phosphate buffer (50 mM, pH 7.0). The particle suspension wasplaced in a vial sealed with a septum and the headspace of the vial wasexchanged for H₂ gas via inlet and outlet needles. Aliquots were removedat specific time intervals for analysis for NAD⁺/NADH content. Eachaliquot was centrifuged to remove particles, removed from the anaerobicglove box, and examined using ultra-violet/visible spectroscopy. NADHgeneration was observed.

Example 2 NAD⁺ Cofactor Regeneration System

All steps were carried out in an anaerobic glove box (Glove BoxTechnology or MBraun) under an atmosphere of N₂. Pyrolytic graphiteparticles modified with Escherichia coli hydrogenase 2 (component (ii))and Ralstonia eutropha diaphorase (HoxFU, component (i)) were preparedas described in Example 1. After collection of the enzyme-modifiedparticles by centrifugation, the particles were resuspended in 1 mM NADHin potassium phosphate buffer (50 mM, pH 7.0). The particle suspensionwas placed in a vial sealed with a septum containing N₂ gas. Aliquotswere removed at specific time intervals for analysis for NAD⁺/NADHcontent. Each aliquot was centrifuged to remove particles and examinedusing ultra-violet/visible spectroscopy. NAD⁺ generation was observed.

Example 3 Preparation of Wild-Type and Variant R. eutropha Diaphorase(HoxFU)

For purification of wild type HoxFU and variants, R. eutropha cellscontaining plasmid pHoxFU harboring the genes hoxFUIhypA2B2F2CDEX (thehoxF gene was equipped at the 3′ end with a sequence encoding theStrep-tag II peptide) were grown heterotrophically in a mineral saltsmedium containing a mixture of 0.2% (w/v) fructose and 0.2% (v/v)glycerol supplemented with 1 μM NiCl₂ and 1 μM ZnCl₂. (Lauterbach et al.PLoS ONE doi:10.1371/journal.pone.0025939). Cells were harvested at anoptical density at 436 nm of 9 to 11 and washed with 50 mM potassiumphosphate (K—PO₄) buffer, pH 7.0 containing 50 mM succinate. Theresulting cell pellet was resuspended in two volumes of resuspensionbuffer (50 mM Tris-HCl, 150 mM KCl, 5% glycerol, pH 8.0 containingProtease Inhibitor (EDTA-free, Roche). After two passages through achilled French pressure cell at 6.2 MPa, the suspension was centrifugedat 100,000×g for 45 min. The soluble extract was applied to a 2 mLStrep-Tactin Superflow column (IBA), washed with 6 mL of resuspensionbuffer and eluted with the same buffer containing 5 mM desthiobiotin(Lauterbach et al. PLoS ONE doi:10.1371/journal.pone.0025939). Fractionscontaining HoxFU protein were pooled, concentrated and subsequently usedfor immobilization on graphite particles.

Diaphorase (HoxFU) variants were isolated as described above except thatthe hoxF sequence on plasmid pHoxFU was altered by genetic engineeringwhich resulted in the production of HoxFU variants containing specificamino acid exchanges, to improve, inter alia, the NADP(H) bindingaffinity.

Example 4 Preparation of Soluble Extract HoxHYFUI2 (SH I64A)

The R. eutropha HF210 strains with the plasmid pGE747 for production ofthe SHI64 derivative were grown heterotrophically in a mineral saltsmedium containing a mixture of 0.2% (w/v) fructose and 0.2% (v/v)glycerol (FGN medium), which were harvested at an optical density at 436nm of 9 to 11. For preparing soluble extract HoxHYFUI2 of the SHI64derivative, the cells were resuspended in two volumes of 50 mm Tris-HCl,150 mm KCl, pH 8.0 buffer containing a protease inhibitor cocktail(EDTA-free Protease Inhibitor, Roche). Cells were broken by two passagesthrough a chilled French pressure cell at 6.2 MPa and the resultingsuspension was centrifuged at 100,000 g for 45 min. The supernatant(soluble extract) was applied for preparing particles.

Example 5 Use of Cofactor Regeneration System of the Present Inventionto Regenerate NADH for a Dehydrogenase

Pyrolytic graphite particles modified with Escherichia coli hydrogenase2 (component (ii)) and Ralstonia eutropha diaphorase (HoxFU, component(i)) were prepared in an anaerobic glove box (Glove Box Technology orMBraun) as described in Example 1. After collection of theenzyme-modified particles by centrifugation, the particles were added toa solution containing S-Lactate dehydrogenase (Sigma, 0.5 mg/mL) andpyruvate (3 mM). To this suspension was added NAD⁺ (0.2 mM), and thesuspension was equilibrated with H₂ gas at atmospheric pressure. Theformation of lactate was detected by Attentuated Total ReflectanceFourier Transform InfraRed spectroscopy using a diamond AttentuatedTotal Reflectance accessory (DurasampIIR II, SensIR Technologies).

Example 6 Use of Cofactor Regeneration System to Supply NADH to anNADH-Dependent Dehydrogenase Enzyme

The product of the dehydrogenase enzyme is a high value fine chemical orpharmaceutical product. A reactor is supplied with particles modifiedwith E. coli hydrogenase 2 (component (ii)) and R. eutropha HoxFU(component (i)). The reactor is also supplied with the dehydrogenaseenzyme, the substrate of the dehydrogenase enzyme (500 mM), NAD⁺ (1 mM),and H₂. After a certain period of time, the product of the dehydrogenasereaction is collected from the reactor(eg by solvent extraction).

Example 7 Regeneration of NADH for Supply to a Dehydrogenase

The cofactor regeneration system of the present invention is placed in asolution of NAD⁺ (eg 0.2 mM) under an atmosphere comprising mainly H₂(eg H₂ gas, or 90% H₂/10% N₂). An NADH-dependent dehydrogenase (eglactate dehydrogenase) is added to the solution (or is attached to theelectronically conducting surface). The substrate for the dehydrogenase(eg pyruvate for lactate dehydrogenase) is placed in the solution. Theproduct of the dehydrogenase reaction (eg S-lactate for S-lactatedehydrogenase) can be collected continuously or batchwise.

Example 8 Regeneration of NADH for Supply to a P450 Monoxygenase

The cofactor regeneration system of the present invention is placed in asolution (free or immobilised on an electrically conducting surface) ofNAD⁺ (eg 0.2 mM) under an atmosphere comprising mainly H₂ with a smallamount of O₂ (eg 99% H₂/1% O₂). An NADH-dependent cytochrome P450mono-oxygenase enzyme is added to the solution (or is attached to theelectronically conducting surface). The substrate for the cytochromeP450 mono-oxygenase is placed in the solution. The product of thecytochrome P450 mono-oxygenase reaction can be collected continuously orbatchwise.

Example 9 Regeneration of NAD⁺ for Supply to a Dehydrogenase

The cofactor regeneration system of the present invention is placed in asolution of NADH (eg 0.2 mM) under an under an inert atmosphere (eg N₂,argon) or an atmosphere containing low level H₂ (eg 1%). AnNAD⁺-dependent dehydrogenase (eg alcohol dehydrogenase) is added to thesolution (or is attached to the electronically conducting surface). Thesubstrate for the dehydrogenase (eg ethanol for alcohol dehydrogenase)is placed in the solution. The product of the dehydrogenase reaction (egacetaldehyde for alcohol dehydrogenase) can be collected continuously orbatchwise.

Example 10 NADPH Cofactor Regeneration System

All steps were carried out in an anaerobic glove box (Glove BoxTechnology or MBraun) under an atmosphere of N2. Particles of pyrolyticgraphite were prepared by abrasion of a piece of pyrolytic graphite withemery paper. These particles were immersed in 100 mM bis-Tris buffer, pH6.0 and suspended by sonication (5 minutes, ultrasonic bath). An aliquotof the particle suspension was removed. To this aliquot was added analiquot of D. vulgaris Miyazaki F hydrogenase (component (ii)) and analiquot of a variant of Ralstonia eutropha diaphorase (HoxHYFU, D467SE341A) (component (i)). The particles and enzymes were left at 4° C. for10 minutes to allow the enzyme components to adsorb onto the particles.Centrifugation (5 minutes, benchtop centrifuge) was used to separate theparticles and to remove excess unadsorbed enzyme. The enzyme-modifiedparticles were then resuspended in 2 mM NADP⁺ in bis-Tris buffer (100mM, pH 6.0). The particle suspension was placed in a vial sealed with aseptum and the headspace of the vial was exchanged for H₂ gas via inletand outlet needles. Aliquots were removed at specific time intervals foranalysis for NADP⁺/NADPH content. Each aliquot was centrifuged to removeparticles, removed from the anaerobic glove box, and examined usingultra-violet/visible spectroscopy. NADPH generation was observed.

Example 11 Use of Cofactor Regeneration System of the Present Inventionto Regenerate NADH for Yeast Alcohol Dehydrogenase

Pyrolytic graphite particles modified with Escherichia coli hydrogenase2 (component (ii)) and a soluble extract of Ralstonia eutrophadiaphorase (HoxHYFU, I64A variant, component (i)) were prepared in ananaerobic glove box (Glove Box Technology or MBraun) according to themethodology described in Example 1. After collection of theenzyme-modified particles by centrifugation, the particles were thenadded to a solution containing yeast alcohol dehydrogenase and heptanal(10 mM). To this suspension was added NAD⁺ (2 mM), and the suspensionwas equilibrated with H₂ gas at atmospheric pressure. Ethyl acetate wasadded to the final solution to extract the product. After thoroughmixing, the organic and aqueous phases were separated by centrifugation.The formation of 1-heptanol in the organic phase was confirmed by gaschromatography detection.

Example 12 NADH Cofactor Regeneration System with Co-Expressed SolubleExtract

Cells from a strain of Ralstonia eutropha H₁₆ incorporating plasmidsencoding the membrane bound hydrogenase and HoxFU are broken open. Themembrane bound hydrogenase is solubilized from the membrane by theaddition of detergent Triton X-100 to crude cell extracts. A subsequenthigh-speed centrifugation step leads to a soluble extract containingboth membrane bound hydrogenase (component (ii)) and HoxFU (component(i)). A soluble cell extract of an Escherichia coli strain withoverexpressed alcohol dehydrogenase is added to the Ralstonia eutrophasoluble extract. Carbon-based particles are added to the soluble extractand left for 30 minutes at 4° C. The particle suspension is warmed to30° C. and the substrate for the alcohol dehydrogenase is added to aconcentration of 500 mM. NAD⁺ is also added to a concentration of 1 mMand H₂ gas is gently bubbled into the solution. After 10 hours theproduct of the alcohol dehydrogenase reaction is collected by solventextraction.

The invention claimed is:
 1. A method for manufacturing a cofactorregeneration system, the method comprising: a. identifying: i. a firstelectron transfer component comprising a NADH: acceptor oxido-reductasethat has activity in an assay which comprises: a. adsorbing the NADH:acceptor oxido-reductase onto an electrode which is immersed in anelectrochemical cell solution containing buffered electrolyte; b.holding the electrode at a constant potential of −412 mV while anincreasing concentration of NAD⁺ is injected into said electrochemicalcell solution; and c. recording a change in the electrocatalytic currentmagnitude at the electrode; or ii. a first electron transfer componentcomprising a NADPH: acceptor oxido-reductase that has activity in anassay which comprises: a. adsorbing the NADPH: acceptor oxido-reductaseonto an electrode which is immersed in an electrochemical cell solutioncontaining buffered electrolyte; b. holding the electrode at a constantpotential of −412 mV while an increasing concentration of NADP+ isinjected into said electrochemical cell solution; and c. recording achange in the electrocatalytic current magnitude at the electrode; andiii. a second electron transfer component comprising a hydrogenasemoiety that has an electrocatalytic H⁺ reduction current,electrocatalytic H₂ oxidation current, or a combination thereof, within100 mV below or above the thermodynamic potential of the 2H⁺/H₂ coupleunder the experimental conditions, when tested in an assay comprising:a. attaching the hydrogenase to an electrode such that the hydrogenasecan exchange electrons directly with the electrode; b. immersing theelectrode in an electrochemical cell solution containing bufferedelectrolyte saturated with H₂; and c. cycling the electrode potentialbetween lower and upper limiting potentials; or iv. a second electrontransfer component comprising a non-biological nanoparticle that has anelectrocatalytic H⁺ reduction current, electrocatalytic H₂ oxidationcurrent, or a combination thereof, within 100 mV below or above thethermodynamic potential of the 2H⁺/H₂ couple under the experimentalconditions, when tested in an assay comprising: a. attaching thenon-biological nanoparticle to an electrode such that the non-biologicalnanoparticle can exchange electrons directly with the electrode; b.immersing the electrode in an electrochemical cell solution containingbuffered electrolyte saturated with H₂; and c. cycling the electrodepotential between lower and upper limiting potentials; and b. providingthe first electron transfer component, the second electron transfercomponent, and an electronically conducting surface; wherein the firstand second electron transfer components do not occur together in natureas an enzyme complex; and c. immobilising the first and second electrontransfer components on the electronically conducting surface so that inuse electrons flow: from the first electron transfer component via theelectronically conducting surface to the second electron transfercomponent; or from the second electron transfer component via theelectronically conducting surface to the first electron transfercomponent.
 2. The method according to claim 1, wherein the firstelectron transfer component is a diaphorase.
 3. The method according toclaim 1, wherein the first electron transfer component comprises anamino acid sequence having at least 90% sequence identity to one or moresequences selected from the group consisting of: SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ IDNO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65;or wherein the second electron transfer component comprises an aminoacid sequence having at least 90% sequence identity to one or moresequences selected from the group consisting of SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34,SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO:53.
 4. A cofactor regeneration system comprising: a. a first electrontransfer component comprising: i) a NADH: acceptor oxido-reductase thathas activity in an assay which comprises: a. adsorbing the NADH:acceptor oxido-reductase onto an electrode which is immersed in anelectrochemical cell solution containing buffered electrolyte; b.holding the electrode at a constant potential of −412 mV while anincreasing concentration of NAD⁺ is injected into said electrochemicalcell solution; and c. recording a change in the electrocatalytic currentmagnitude at the electrode; or ii) a NADPH acceptor: oxido-reductasethat has activity in an assay which comprises: a. adsorbing the NADPH:acceptor oxido-reductase onto an electrode which is immersed in anelectrochemical cell solution containing buffered electrolyte; b.holding the electrode at a constant potential of −412 mV while anincreasing concentration of NADP⁺ is injected into said electrochemicalcell solution; and c. recording a change in the electrocatalytic currentmagnitude at the electrode; and b. a second electron transfer componentcomprising: i) a hydrogenase moiety that has an electrocatalytic H⁺reduction current, electrocatalytic H₂ oxidation current, or acombination thereof, within 100 mV below or above the thermodynamicpotential of the 2H⁺/H₂ couple under the experimental conditions, whentested in an assay comprising: a. attaching the hydrogenase to anelectrode such that the hydrogenase can exchange electrons directly withthe electrode; b. immersing the electrode in an electrochemical cellsolution containing buffered electrolyte saturated with H₂; and c.cycling the electrode potential between lower and upper limitingpotentials; or ii) a non-biological nanoparticle that has anelectrocatalytic H⁺ reduction current, electrocatalytic H₂ oxidationcurrent, or a combination thereof, within 100 mV below or above thethermodynamic potential of the 2H⁺/H₂ couple under the experimentalconditions, when tested in an assay comprising: a. attaching thenon-biological nanoparticle to an electrode such that the non-biologicalnanoparticle can exchange electrons directly with the electrode; b.immersing the electrode in an electrochemical cell solution containingbuffered electrolyte saturated with H₂; and c. cycling the electrodepotential between lower and upper limiting potentials; and c. anelectronically conducting surface; and wherein the first and secondelectron transfer components are immobilised on the electronicallyconducting surface; and wherein the first and second electron transfercomponents do not occur together in nature as an enzyme complex; andwherein the cofactor regeneration system is configured so that in useelectrons flow: from the first electron transfer component via theelectronically conducting surface to the second electron transfercomponent; or from the second electron transfer component via theelectronically conducting surface to the first electron transfercomponent.
 5. The method according to claim 1, wherein the firstelectron transfer component comprises an amino acid sequence having atleast 90% sequence identity to one or more sequences selected from thegroup consisting of: SEQ ID NO: 66 and SEQ ID NO: 67; or wherein thesecond electron transfer component comprises an amino acid sequencehaving at least 90% sequence identity to one or more sequences selectedfrom the group consisting of: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, and SEQ ID NO: 72.